The platelet membrane is involved in many aspects of the platelet's role in hemostasis and thrombosis: adhesion, aggregation, thrombin generation on the platelet surface, interaction with polymerizing fibrin, and clot retraction. The objectives of my research are to define (1) the transbilayer organization of platelet membrane proteins, (2) the membrane changes which occur with platelet senescence in normal subjects, (3) platelet membrane abnormalities associated with thrombotic and hemorrhagic disease, and (4) membrane protein alterations associated with in vitro activation. Our previous studies have developed quantitative methods for complete isolation of whole blood platelets and the assessment of platelet glycoproteins by surface radioisotope labeling with (125I)-diazotized diiodosulfanilic acid (DD125ISA) and planimetry of densitometer scans of periodic acid-Schiff (PAS)-stained SDS-polyacrylamide gels (SDS-PAGE). We have isolated and immunologically identified platelet microparticles in cell-free plasma. We will selectively isolate plasma membranes on polylysine-coated acrylamide beads and define the trans-bilayer organization of membrane proteins of normal and activated platelets. Using these methods we will determine the membrane changes associated with human platelet senescence, since surface glycoproteins may be diminished in older platelets. We will define the platelet membrane changes in thrombotic disease: Are platelet surface changes present which are similar to the result of in vitro activation? Are circulating microparticles increased in the plasma? We will also extend our observations on the membrane changes which occur with platelet secretion, to immunologically confirm the appearance of actin on the platelet surface, define the mechanism of this change, and study the function of platelet surface actin.